METHODS FOR DIAGNOSING LHRH OR hCG/LH RECEPTOR EXPRESSING TUMORS, CANCERS AND NEOPLASIAS

ABSTRACT

The invention relates to diagnosis, detection, screening, identifying and predicting methods. In various embodiments, methods of the invention include diagnosis, detection, or screening for a hyperproliferative disorder (e.g., a tumor, cancer or neoplasia) in the subject; identifying a subject that will or is likely to respond to a therapy for a hyperproliferative disorder (e.g., a tumor, cancer or neoplasia); and predicting therapeutic efficacy of a hyperproliferative disorder (e.g., a tumor, cancer or neoplasia) treatment in a subject.

RELATED APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 16/154,486, filed Oct. 8, 2018, which is a continuation of U.S.patent application Ser. No. 12/975,058, filed Dec. 21, 2010, now U.S.Pat. No. 10,093,961, issued Oct. 9, 2018, which claims priority to U.S.Provisional Patent Application No. 61/289,010, filed Dec. 22, 2009. Theentire contents of the foregoing applications are incorporated herein byreference, including all text, tables, sequence listings and drawings.

TECHNICAL FIELD

The invention relates to diagnosis, detection, screening, identifyingand predicting methods. In various embodiments, methods of the inventioninclude diagnosis, detection, screening for or imaging ahyperproliferative disorder (e.g., a tumor, cancer or neoplasia) in thesubject; identifying a subject that will or is likely to respond totherapy for a hyperproliferative disorder (e.g., a tumor, cancer orneoplasia, or a metastasis thereof); and predicting therapeutic efficacyof a treatment for a hyperproliferative disorder (e.g., a tumor, canceror neoplasia, or a metastasis thereof) in a subject.

Introduction

The need to develop new diagnosis, detection, screening, imaging,identification and predictive methods for hyperproliferative disorders(e.g., a tumor, cancer or neoplasia, or a metastasis thereof) isevident. For example, the five year survival rate is only 10-40% forpatients with lung, colorectal, breast and prostate cancer if diagnosedwith distant metastatic disease.

SUMMARY

The invention provides methods and kits for diagnosis, detection,screening and imaging, in vitro, ex vivo and in vivo. Methods include,among other things, contacting a sample (e.g., a biological sample) fromor of a subject with an agent that detects the presence of one or morehormone receptors (e.g., LHRH- or hCG/LH receptors) in the sample, andcorrelating the amount or expression of hormone receptors (e.g., LHRH-or hCG/LH receptors) with the presence of or increased risk of having ahyperproliferative disorder (e.g., a tumor, cancer or neoplasia, or ametastasis thereof), thereby identifying the subject as having or atrisk of having a hyperproliferative disorder (e.g., a tumor, cancer orneoplasia, or a metastasis thereof). Methods also include, among otherthings, administering an agent (e.g., a detectable or labeled agent) toa subject having or at risk of having a hyperproliferative disorder(e.g., a tumor, cancer or neoplasia, or a metastasis thereof) in anamount effective to diagnose, detect, screen for or image ahyperproliferative disorder (e.g., a tumor, cancer or neoplasia, or ametastasis thereof) in the subject. In particular aspects, a sample or asubject in which at least 1-25% of the cells express a hormone receptor(e.g., LHRH- or hCG/LH receptors) correlates with the presence or anincreased risk of a hyperproliferative disorder (e.g., a tumor, canceror neoplasia, or a metastasis thereof), or cells in the sample orsubject express more of a hormone receptor (e.g., LHRH- or hCG/LHreceptors), for example, the cells in the sample express at least 10%,25%, 30%, 40%, 45%, or 50% or more of the hormone receptor thancomparative control cells (e.g., normal cells), which correlates withthe presence or an increased risk of a hyperproliferative disorder(e.g., a tumor, cancer or neoplasia, or a metastasis thereof) in thesubject.

The invention also provides methods and kits for identifying a subjectthat will or is likely to respond to a treatment or a therapy thattargets a receptor (e.g., a hormone receptor) or that targets ahyperproliferative disorder (e.g., a tumor, cancer or neoplasia, or ametastasis thereof), in vitro, ex vivo and in vivo. In one embodiment, amethod includes, contacting a biological sample from a subject with anagent that detects the presence of a hormone receptor—the presence thehormone receptor (e.g., LHRH- or hCG/LH receptors) in the sampleindicates that the subject will or is likely to respond to a treatmentor therapy (e.g., for a hyperproliferative disorder such as a tumor,cancer or neoplasia, or a metastasis thereof) that targets the hormonereceptor. In another embodiment, a method includes, administering anagent that detects the presence of a hormone receptor to a subject—thepresence of the hormone receptor (e.g., LHRH- or hCG/LH receptors) inthe subject indicates that the subject will or is likely to respond to atreatment or therapy (e.g., for a hyperproliferative disorder such as atumor, cancer or neoplasia, or a metastasis thereof) that targets thehormone receptor.

The invention further provides in vitro, ex vivo and in vivo methods andkits for predicting therapeutic efficacy for a treatment or therapy thattargets a receptor (e.g., a hormone receptor) or that targets ahyperproliferative disorder (e.g., a tumor, cancer or neoplasia, or ametastasis thereof). Methods include, among other things, contacting abiological sample from a subject with an agent that detects the presenceof a hormone receptor, and correlating the amount of hormone receptor(e.g., LHRH- or hCG/LH receptors) present with the likelihood oftherapeutic efficacy of treatment or therapy that targets the hormonereceptor (e.g., LHRH- or hCG/LH receptors), thereby predicting thetherapeutic efficacy of the treatment or therapy that targets thehormone receptor. In particular aspects, hormone receptor amounts in thesample greater than normal, such as hormone expressing cells in thesample or subject expressing more of a hormone receptor than istypically present in an appropriate control sample (e.g., normal cells),is predictive of therapeutic efficacy, for example, when 1% or more ofthe cells in the sample (e.g., 5%, 10%, 15%, 20%, 25%, 30%, etc. ormore) express a hormone receptor, which is predictive of therapeuticefficacy for treatment that targets a hormone receptor or that targets ahyperproliferative disorder (e.g., a tumor, cancer or neoplasia, or ametastasis thereof).

Biological samples analyzed in accordance with the methods of theinvention, and subjects treatable in accordance with the methods of theinvention include, for example, an animal such as a mammal cells, or acell, tissue or organ biopsy. In particular embodiments, a biologicalsample analyzed or a subject administered an agent is of an animal, suchas a mammal (e.g., a human). Non-limiting biological sample can be froma reproductive cell, tissue or organ, such as breast, endometrium,uterus, ovary, testes, and other cells, tissues and organs, such asprostate, colon, pancreas, esophagus, liver, skin, kidney, adrenalgland, brain and blood.

Therapeutic efficacy for a treatment or therapy that targets a receptor(e.g., a hormone receptor) include, for example, a polypeptide thatbinds to the receptor, such as an antibody or receptor ligand, or apolynucleotide that hybridizes to a nucleic acid encoding all or aportion of a receptor. In particular embodiments, an agent includes apolypeptide that binds to an LHRH- or hCG/LH receptor, or apolynucleotide that hybridizes to a nucleic acid sequence encoding allor a portion of an LHRH- or hCG/LH receptor. Non-limiting examples ofligands include, for LHRH receptor for example, gonadotropin-releasinghormone I, gonadotropin-releasing hormone II, lamprey III luteinizinghormone releasing hormone, and fragments thereof; and for hCG/LHreceptor, ligands include luteinizing hormone beta chain, luteinizinghormone (LH), chorionic gonadotropin (CG), chorionic gonadotropin betasubunit (β- or beta-CG), and fragments thereof. Non-limiting examples ofagents that bind to a receptor include, for LHRH receptor for example,leuprolide, leuprolide acetate (Lupron™), Goserelin (Zoladex™),Histrelin (Supprelin™), Triptorelin (Trelstar™), Buserelin (Suprefact™),Cetrorelix (Cetrotide™), Ganirelix (Antagon™), Antide, Abarelix(Plenaxis™), Teverelix (Antarelix™), Fe 200486 (Degarelix), Nal-Glu orElagolix (NBI-56418) or an analog thereof.

Detection, diagnostic and imaging methods include, but are not limitedto immunohistochemistry (IHC), in-situ hybridization (ISH), ELISA,immunoprecipitation, immunofluorescence, chemiluminescence,radioactivity, X-ray, nucleic acid hybridization, and protein-proteininteraction. Detection, diagnostic and imaging methods also include, butare not limited to immunoprecipitation, ELISA, flow cytometry, Westernblotting, polymerase chain reaction, DNA transcription, Northernblotting and Southern blotting.

Tumors, cancers and neoplasias can be present or affect and therefore bedetected, diagnosed or imaged in breast, endometrium, uterus, ovary,testes, lung, prostate, colon, pancreas, esophagus, liver, skin, kidney,adrenal gland, brain, and which therefore include detection, diagnosisand imaging of lymphoma, breast, endometrium, uterus, ovary, testes,lung, prostate, colon, pancreas, esophagus, liver, skin, kidney, adrenalgland, and brain tumors, cancers and neoplasias. Tumors, cancers andneoplasias can be a metastasis or recurring tumor, cancer or neoplasia.

DESCRIPTION OF THE DRAWINGS

FIG. 1: Shows LHRH receptors in biopsies from breast cancer patientsstained with anti-LHRH receptor monoclonal antibody, clone A9E4. Eachpanel shows the intensity of expression evaluated through the VentanaImage Analysis System (VIAS) adjunctive computer assisted image analysissystem functionally connected to an interactive microscope (AxioImager).

FIGS. 2A and 2B: Human cancer cell lines show high correlation tosensitivity to EP-100 (KFAKFAKKFAKFAKKFAKQHWSYGLRPG) (SEQ ID NO.:1) andtheir presence levels of functional LHRH receptors determined asreceptor capacities and VIAS Scores. Human cancer cell lines wereincubated with EP-100 for 24 h showed variable sensitivities to thedrug, which correlated with LHRH receptor capacities among those twomulti-drug resistant cells.

DETAILED DESCRIPTION

In accordance with the invention, there are provided methods and kitsfor diagnosis, detection, screening and imaging of a hyperproliferativedisorder (e.g., a tumor, cancer or neoplasia, or metastasis thereof) invitro, ex vivo and in vivo. In one embodiment, a method of the inventionincludes administering an agent to the subject under conditions wherebythe agent can bind to a hyperproliferative cell (e.g., a tumor, canceror neoplasia, or metastasis thereof), and detecting the agent in thesubject to diagnose, detect, screen or image a hyperproliferativedisorder (e.g., a tumor, cancer or neoplasia, or metastasis thereof) inthe subject. In another embodiment, a method of the invention includesadministering an agent to the subject under conditions whereby the agentcan bind to a hyperproliferative cell (e.g., a tumor, cancer orneoplasia, or metastasis thereof), and detecting the agent in thesubject to ascertain the presence or absence of hyperproliferative cells(e.g., a tumor, cancer or neoplasia, or metastasis thereof), therebydetecting hyperproliferative cells (e.g., a tumor, cancer or neoplasia,or metastasis thereof), or diagnosing the subject as having or nothaving hyperproliferative cells (e.g., a tumor, cancer or neoplasia, ormetastasis thereof). In an additional embodiment, a method of theinvention includes administering an agent to the subject underconditions whereby the agent can bind to hyperproliferative cells (e.g.,a tumor, cancer or neoplasia, or metastasis thereof), and imaging theagent in the subject to ascertain the presence or absence, or the extent(progression or regression) or location of the hyperproliferative cells(e.g., a tumor, cancer or neoplasia, or metastasis thereof) in thesubject.

Invention methods include, among other things, in vitro, ex vivo and invivo contact and/or administration. A sample, such a biological sample,can be contacted with, administered, or delivered an agent in order toeffect a method of the invention, for example, to diagnose, detect,screen for or to image a hyperproliferative disorder (e.g., a tumor,cancer or neoplasia, or metastasis thereof), to predict therapeuticefficacy of a treatment for a hyperproliferative disorder (e.g., atumor, cancer or neoplasia, or metastasis thereof), or to predicttherapeutic efficacy of a treatment that targets a receptor (e.g.,hormone receptor). Subjects can also be contacted with, administered, ordelivered an agent in order to effect a method of the invention, forexample, to diagnose, detect, screen for or image a hyperproliferativedisorder (e.g., a tumor, cancer or neoplasia, or metastasis thereof), topredict therapeutic efficacy of a treatment that targets a receptor(e.g., hormone receptor), or to predict therapeutic efficacy of atreatment for a hyperproliferative disorder (e.g., a tumor, cancer orneoplasia, or metastasis thereof).

The term “contact” and grammatical variations thereof means a sample ora subject is given or delivered an agent under conditions allowing aphysical interaction (direct or indirect) between an agent and areceptor, such as a receptor expressed on a hyperproliferative cell(e.g., a tumor, cancer or neoplasia, or metastasis thereof), or ahyperproliferative cell, which may be present in the sample or in asubject. Contact as used herein includes in solution, in solid phase, invitro, ex vivo, in a cell and in vivo. Thus, methods of the inventioninclude contact of an agent with a target, such as a receptor (e.g., ahormone receptor) or cell (e.g., a hyperproliferative cell), underconditions allowing the agent to bind to the receptor or cell, if thereceptor or cell are present in a sample or in a subject. The term“administering” includes delivery to a subject in which the agent cancontact (e.g., physically interact, directly or indirectly) with areceptor (e.g., a hormone receptor) such as a receptor expressed on ahyperproliferative cell (e.g., a tumor, cancer or neoplasia, ormetastasis thereof), or a hyperproliferative cell, in vivo, i.e., in thesubject.

In particular embodiments of the invention, an agent is a molecule thatbinds, under appropriate conditions, to a receptor, such as a hormonereceptor (e.g., expressed on a hyperproliferative cell), or to ahyperproliferative cell (e.g., a tumor, cancer or neoplasia, ormetastasis thereof) and that is detectable. The term “bind,” or“binding,” when used in reference to an agent, means that the agentphysically interacts (directly or indirectly) at the molecular levelwith a receptor protein or a receptor encoding nucleic acid sequence.Thus, an agent can bind to all or a part of receptor protein or nucleicacid sequence. Typically, binding is that which is specific or selectivefor the receptor or nucleic acid. Specific and selective binding can bedistinguished from non-specific binding using assays known in the art(e.g., for protein detection, immunoprecipitation, ELISA, flowcytometry, and Western blotting, and for nucleic acid detection,polymerase chain reaction, DNA transcription, northern and southernblotting, etc.).

Agents include organic and inorganic molecules. Organic agents include,for example, polypeptide and nucleic acid sequences, and small organicligands. Inorganic agents include, for example, metals, metal oxides,radioactive isotopes, fluorophores, chromophores, and electron-densereagents.

As used herein, the terms “polypeptide” “protein,” “peptide” and “aminoacid sequence” are used interchangeably herein to refer to two or moreamino acids, or “residues,” covalently linked by an amide bond orequivalent. Residues of amino acid sequences can be linked by naturalamide bonds, or by non-natural or non-amide chemical bonds.

Polypeptide agents include ligands, for example, a ligand or antibodythat binds to a receptor (e.g., a hormone receptor such as LHRH- orhCG/LH receptors). Ligands also include hormones, which bind to hormonereceptors include, for example, mammalian forms, such as primate (e.g.,human) hormones.

Non-limiting exemplary receptor ligands for LHRH receptors, includegonadotropin-releasing hormone I, gonadotropin-releasing hormone II,lamprey III luteinizing hormone releasing hormone, and fragments thereofthat bind to LHRH receptors. Non-limiting exemplary receptor ligands forhCG/LH include luteinizing hormone beta chain, luteinizing hormone (LH),chorionic gonadotropin (CG), chorionic gonadotropin beta subunit (β- orbeta-CG), or fragments thereof that bind to hCG/LH receptors (e.g.,chorionic gonadotropin beta subunit fragment 81-95).

Representative hormone receptors include, for example, LHRH (luteinizinghormone releasing hormone, aka gonadotropin-releasing hormone) receptor,and CG (chorionic gonadotropin hormone, aka luteinizing hormone)receptor. Hormone receptors include, for example, mammalian forms, suchas primate (e.g., human) hormone receptors.

A representative human LHRH receptor sequence includes, but is notlimited to, full length or a subsequence of (SEQ ID NO.:2):

1 MANSASPEQN QNHCSAINNS IPLMQGNLPT LTLSGKIRVT VTFFLFLLSA TFNASFLLKL 61QKWTQKKEKG KKLSRMKLLL KHLTLANLLE TLIVMPLDGM WNITVQWYAG ELLCKVLSYL 121KLFSMYAPAF MMVVISLDRS LAITRPLALK SNSKVGQSMV GLAWILSSVF AGPQLYIFRM 181IHLADSSGQT KVFSQCVTHC SFSQWWHQAF YNFFTFSCLF IIPLFIMLIC NAKIIFTLTR 241VLHQDPHELQ LNQSKNNIPR ARLKTLKMTV AFATSFTVCW TPYYVLGIWY WFDPEMLNRL 301SDPVNHFFFL FAFLNPCFDP LIYGYFSL.

A representative hCG/LH human receptor sequence includes, but is notlimited to, full length or a subsequence of (SEQ ID NO.: 3):

1 MKQRFSALQL LKLLLLLQPP LPRALREALC PEPCNCVPDG ALRCPGPTAG LTRLSLAYLP 61VKVIPSQAFR GLNEVIKIEI SQIDSLERIE ANAFDNLLNL SEILIQNTKN LRYIEPGAFI 121NLPRLKYLSI CNTGIRKFPD VTKVFSSESN FILEICDNLH ITTIPGNAFQ GMNNESVTLK 181LYGNGFEEVQ SHAFNGTTLT SLELKENVHL EKMHNGAFRG ATGPKTLDIS STKLQALPSY 241GLESIQRLIA TSSYSLKKLP SRETFVNLLE ATLTYPSHCC AFRNLPTKEQ NFSHSISENF 301SKQCESTVRK VSNKTLYSSM LAESELSGWD YEYGFCLPKT PRCAPEPDAF NPCEDIMGYD 361FLRVLIWLIN ILAIMGNMTV LFVLLTSRYK LTVPRFLMCN LSFADFCMGL YLLLIASVDS 421QTKGQYYNHA IDWQTGSGCS TAGFFTVFAS ELSVYTLTVI TLERWHTITY AIHLDQKLRL 481RHAILIMLGG WLFSSLIAML PLVGVSNYMK VSICFPMDVE TTLSQVYILT ILILNVVAFF 541IICACYIKIY FAVRNPELMA TNKDTKIAKK MAILIFTDFT CMAPISFFAI SAAFKVPLIT 601VTNSKVLLVL FYPINSCANP FLYAIFTKTF QRDFFLLLSK FGCCKRRAEL YRRKDFSAYT 661SNCKNGFTGS NKPSQSTLKL STLHCQGTAL LDKTRYTEC.

Agents therefore include molecules that bind to hormone receptors, whichinclude hormones and hormone subsequences. Non-limiting representativehormones include, for example, gonadotropin-releasing hormone I,gonadotropin-releasing hormone II, and lamprey III luteinizing hormonereleasing hormone. Representative hormones also include, for example,luteinizing hormone (LH), luteinizing hormone beta chain, chorionicgonadotropin (CG), chorionic gonadotropin beta chain, folliclestimulating hormone (FSH), follicle stimulating hormone (FSH) betachain, thyroid stimulating hormone (TSH), and thyroid stimulatinghormone (TSH) beta chain. A representative subsequence is chorionicgonadotropin beta subunit, fragment 81-95.

The human luteinizing hormone (LH), chorionic gonadotropin (CG),follicle stimulating hormone (FSH), and thyroid stimulating hormone(TSH) are dimers consisting of alpha and beta subunits which associatenoncovalently. The alpha subunits of these hormones are all the same,but their beta chains are different and confer specificity.

A representative human gonadotropin-releasing hormone I sequenceincludes, but is not limited to, full length or a subsequence of (SEQ IDNO.:4):

1 pGlu (E) HWSYGLRPG

A representative human gonadotropin-releasing hormone II sequenceincludes, but is not limited to, full length or a subsequence of (SEQ IDNO.: 5):

1 pEHWSHGWYPG

A representative lamprey III luteinizing hormone releasing hormonesequence includes, but is not limited to, full length or a subsequenceof (SEQ ID NO.: 6): pEHWSHDWKPG.

A representative luteinizing hormone (LH), chorionic gonadotrophin (CG),follicle stimulating hormone (FSH), and thyroid stimulating hormone(TSH) alpha subunit (precursor) sequence includes, but is not limitedto, full length or a subsequence of (SEQ ID NO.: 7):

MDYYRKYAAI FLVTLSVFLH VLHSAPDVQD CPECTLQENPFFSQPGAPIL QCMGCCFSRA YPTPLRSKKT MLVQKNVTSESTCCVAKSYN RVTVMGGFKV ENHTACHCST CYYHKS

A representative luteinizing hormone (LH) beta sequence includes, but isnot limited to, full length or a subsequence of (SEQ ID NO.: 8):

SREPLRPWCH PINAILAVEK EGCPVCITVN TTICAGYCPTMMRVLQAVLP PLPQVVCTYR DVRFESIRLP GCPRGVDPVVSFPVALSCRC GPCRRSTSDC GGPKDHPLTC DHPQLSGLLF L.

A representative chorionic gonadotrophin (CG) beta sequence includes,but is not limited to, full length or a subsequence (e.g., fragment81-95, SYAVALSCQCALARR) of (SEQ ID NO.: 9):

1 SKEPLRPRCR PINATLAVEK EGCPVCITVN TTICAGYCPT 61MTRVLQGVLP ALPQVVCNYR DVRFESIRLP GCPRGVNPVV SYAVALSCQC ALCRRSTTDC 121GGPKDHPLTC DDPRFQDSSS SKAPPPSLPS PSRLPGPSDT PILPQ.

Polypeptide agents further include antibodies. As used herein the term“antibody” refers to a protein that binds to other molecules (antigens)via heavy and light chain variable domains, V_(H) and V_(L),respectively. Antibodies include full-length antibodies that include twoheavy and two light chain sequences. Antibodies can have kappa or lambdalight chain sequences, either full length as in naturally occurringantibodies, mixtures thereof (i.e., fusions of kappa and lambda chainsequences), and subsequences/fragments thereof.

Antibodies include monoclonal and polyclonal immunoglobulin moleculesthat belong to any class such as IgM, IgG, IgA, IgE, IgD, and anysubclass thereof. Exemplary subclasses for IgG are IgG₁, IgG₂, IgG₃ andIgG₄.

A “monoclonal” antibody refers to an antibody that is based upon,obtained from or derived from a single clone, including any eukaryotic,prokaryotic, or phage clone. A “monoclonal” antibody is thereforedefined structurally, and not the method by which it is produced.

Antibodies also include subsequences and fragments that bind to thereceptor or hyperproliferative cell. Antibody subsequences andfragments, including single-chain antibodies, can include all or aportion of heavy or light chain variable region sequences (e.g., CDR1,CDR2 or CDR3 in a heavy chain variable region sequence or in a lightchain variable region sequence) alone or in combination with all or aportion of one or more of the following: hinge region, CH1, CH2, and CH3domains. Non-limiting representative subsequences and fragments of anantibody include but are not limited to Fab, Fab′, F(ab′)₂, Fv, Fd,single-chain Fv (scFv), disulfide-linked Fvs (sdFv), V_(L), V_(H),trispecific (Fab₃), bispecific (Fab₂), diabody ((V_(L)-V_(H))₂ or(V_(H)-V_(L))₂), triabody (trivalent), tetrabody (tetravalent), minibody((scFv-C_(H)3)₂), bispecific single-chain Fv (Bis-scFv), IgGdeltaCH2,scFv-Fc, (scFv)₂-Fc and IgG4PE.

Antibodies include mammalian, primatized, humanized, fully humanantibodies and chimeras. A mammalian antibody is an antibody which isproduced by a mammal, transgenic or non-transgenic, or a non-mammalianorganism engineered to produce a mammalian antibody, such as anon-mammalian cell (bacteria, yeast, insect cell), animal or plant.

A “human” antibody means that the amino acid sequence of the antibody isfully human, i.e., human heavy and human light chain variable and humanconstant regions. Thus, all of the amino acids are human or exist in ahuman antibody. An antibody that is non-human may be made fully human bysubstituting the non-human amino acid residues with amino acid residuesthat exist in a human antibody Amino acid residues present in humanantibodies, CDR region maps and human antibody consensus residues areknown in the art (see, e.g., Kabat, Sequences of Proteins ofImmunological Interest, 4^(th) Ed. US Department of Health and HumanServices. Public Health Service (1987); Chothia and Lesk (1987). Aconsensus sequence of human V_(H) subgroup III, based on a survey of 22known human V_(H) III sequences, and a consensus sequence of human V_(L)kappa-chain subgroup I, based on a survey of 30 known human kappa Isequences is described in Padlan Mol. Immunol. 31:169 (1994); and PadlanMol. Immunol. 28:489 (1991). Human antibodies therefore includeantibodies in which one or more amino acid residues have beensubstituted with one or more amino acids present in any other humanantibody.

A “humanized” antibody, means that the amino acid sequence of theantibody has non-human amino acid residues (e.g., mouse, rat, goat,rabbit, etc.) of one or more complementarity determining regions (CDRs)that specifically bind to the desired antigen in an acceptor humanimmunoglobulin molecule, and one or more human amino acid residues inthe Fv framework region (FR), which are amino acid residues that flankthe CDRs. Such antibodies typically have reduced immunogenicity andtherefore a longer half-life in humans as compared to the non-humanparent antibody from which one or more CDRs were obtained or are basedupon.

Antibodies include those referred to as “primatized,” which are“humanized” except that the acceptor human immunoglobulin molecule andframework region amino acid residues may be any primate amino acidresidue (e.g., ape, gibbon, gorilla, chimpanzees orangutan, macaque), inaddition to any human residue. Human FR residues of the immunoglobulincan be replaced with corresponding non-human residues. Residues in theCDR or human framework regions can therefore be substituted with acorresponding residue from the non-human CDR or framework region donorantibody to alter, generally to improve, antigen affinity orspecificity, for example. A humanized antibody may include residues,which are found neither in the human antibody nor in the donor CDR orframework sequences. For example, a FR substitution at a particularposition that is not found in a human antibody or the donor non-humanantibody may be predicted to improve binding affinity or specificityhuman antibody at that position. Antibody framework and CDRsubstitutions based upon molecular modeling are well known in the art,e.g., by modeling of the interactions of the CDR and framework residuesto identify framework residues important for antigen binding andsequence comparison to identify unusual framework residues at particularpositions (see, e.g., U.S. Pat. No. 5,585,089; and Riechmann et al.,Nature 332:323 (1988)).

A “chimeric” antibody, means that the amino acid sequence of theantibody contains one or more portions that are derived from, obtainedor isolated from, or based upon two or more different species. Forexample, a portion of the antibody may be human (e.g., a constantregion) and another portion of the antibody may be non-human (e.g., amurine heavy or murine light chain variable region). Thus, an example ofa chimeric antibody is an antibody in which different portions of theantibody are of different species origins. Unlike a humanized orprimatized antibody, a chimeric antibody can have the different speciessequences.

Organic agents further include small organic ligands. Non-limitingexemplary small organic ligand agents include, for LHRH receptors,leuprolide, leuprolide acetate (Lupron™), Goserelin (Zoladex™),Histrelin (Supprelin™) Triptorelin (Trelstar™), Buserelin (Suprefact™),Cetrorelix (Cetrotide™), Ganirelix (Antagon™), Antide, Abarelix(Plenaxis™), Teverelix (Antarelix™), Fe 200486 (Degarelix), Nal-Glu andElagolix (NBI-56418).

Organic agents also include nucleic acid sequences. As used herein, theterms “nucleic acid” and “polynucleotide” and the like refer to at leasttwo or more ribo- or deoxy-ribonucleic acid base pairs (nucleotides)that are linked through a phosphoester bond or equivalent covalent bond.Nucleic acids include polynucleotides and polynucleosides. Nucleic acidsinclude single, double or triplex, circular or linear, molecules.Exemplary nucleic acids include but are not limited to: RNA, DNA, cDNA,genomic nucleic acid, naturally occurring and non naturally occurringnucleic acid, e.g., synthetic nucleic acid.

Nucleic acid agents include, for example, polynucleotides thathybridizes to a nucleic acid sequence encoding all or a portion of areceptor (e.g., a hormone receptor such as LHRH- or hCG/LH receptors).As representative protein sequences for various hormone receptors andhormones are disclosed herein or known to the skilled artisan, nucleicacids that hybridize to such sequences can be used in the methods of theinvention for detecting receptors, including hormone receptors.

In order to detect a receptor, a nucleic acid can term “hybridize” toall or a portion of the receptor encoding nucleic acid, which refers tothe binding between two or more nucleic acid sequences. Hybridizingsequences will generally be more than about 50% complementary to all ora portion of a nucleic acid that encodes the receptor. The hybridizationregion between hybridizing sequences typically is at least about 10-15nucleotides, 15-20 nucleotides, 20-30 nucleotides, 30-50 nucleotides,50-100 nucleotides, 100 to 200 nucleotides or more, or any numericalvalue or range within or encompassing such lengths.

The term “complementary” or “antisense” refers to a polynucleotide orpeptide nucleic acid (PNA) capable of binding to a specific DNA or RNAsequence. Antisense includes single, double, triple or greater strandedRNA and DNA polynucleotides and peptide nucleic acids (PNAs) that bindRNA transcript or DNA. Particular examples include RNA and DNA antisensethat binds to sense RNA. For example, a single stranded nucleic acid cantarget a transcript that encodes a receptor. Antisense molecules aretypically 90-100% complementary to the sense strand but can be“partially” complementary, in which only some of the nucleotides bind tothe sense molecule (less than 100% complementary, e.g., 95%, 90%, 80%,70% and sometimes less), or any numerical value or range within orencompassing such percent values.

Nucleic acids can be of various lengths. Nucleic acid lengths typicallyrange from about 10 nucleotides to 20 Kb, or any numerical value orrange within or encompassing such lengths, e.g., 10 nucleotides to 10Kb, 1 to 5 Kb or less, 1000 to about 500 nucleotides or less in length.Nucleic acids can also be shorter, for example, 100 to about 500nucleotides, or from about 10 to 25, 25 to 50, 50 to 100, 100 to 250, orabout 250 to 500 nucleotides in length, or any numerical value or rangeor value within or encompassing such lengths. In particular aspects, anucleic acid sequence has a length from about 10-20, 20-30, 30-50,50-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-1000,1000-2000, nucleotides, or any numerical value or range within orencompassing such lengths. Shorter polynucleotides are commonly referredto as “oligonucleotides” or “probes” of single- or double-stranded DNA.However, there is no upper limit to the length of such oligonucleotides.

Nucleic acid sequences can include nucleotide and nucleosidesubstitutions, additions and deletions, as well as derivatized forms andfusion/chimeric sequences (e.g., encoding recombinant polypeptide). Forexample, due to the degeneracy of the genetic code, nucleic acidsinclude sequences and subsequences degenerate with respect to nucleicacids that encode a receptor and subsequences thereof, such as thehormone receptors set forth herein, as well as variants andmodifications thereof (e.g., substitutions, additions insertions anddeletions).

Nucleic acids can be produced using various standard cloning andchemical synthesis techniques. Techniques include, but are not limitedto nucleic acid amplification, e.g., polymerase chain reaction (PCR),with genomic DNA or cDNA targets using primers (e.g., a degenerateprimer mixture) capable of annealing to antibody encoding sequence.Nucleic acids can also be produced by chemical synthesis (e.g., solidphase phosphoramidite synthesis) or transcription from a gene. Thesequences produced can then be translated in vitro, or cloned into aplasmid and propagated and then expressed in a cell (e.g., a host cellsuch as eukaryote or mammalian cell, yeast or bacteria, in an animal orin a plant).

Agents include modifications or variations, such as substitutions,additions and deletions as long as such modifications, variations,substitutions, additions and deletions do not destroy activity, e.g.,binding activity. Such agents therefore include subsequences and aminoacid and nucleic acid substitutions of the sequences set forth herein,such as a polypeptide agent (e.g., a hormone) that binds to a target(e.g., receptor), or a nucleic acid agent that binds to a target nucleicacid sequence that encodes a receptor (e.g., a hormone receptor). Inparticular embodiments, a subsequence of an agent has 5 to 10, 10 to 15,15 to 20, 20 to 25, 25 to 30, 30 to 35 or more amino acid or nucleotideresidues.

Specific examples include a substitution or deletion of one or moreamino acid (e.g., 1-3, 3-5, 5-10, 10-20, or more) residues of apolypeptide. A “conservative amino acid substitution” is a replacementof one amino acid by a biologically, chemically or structurally similarresidue. Biologically similar means that the substitution is compatiblewith a biological activity, e.g., binding activity. Structurally similarmeans that the amino acids have side chains with similar length, such asalanine, glycine and serine, or having similar size, or a structure ismaintained. Chemical similarity means that the residues have the samecharge or are both hydrophilic or hydrophobic. Particular examplesinclude the substitution of one hydrophobic residue, such as isoleucine,valine, leucine or methionine for another, or the substitution of onepolar residue for another, such as the substitution of arginine forlysine, glutamic for aspartic acids, or glutamine for asparagine, serinefor threonine, etc.

A modified agent, such as a hormone, can have a sequence with 50%, 60%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or more identity to areference sequence (e.g., a hormone sequence). Due to variation in theamount of sequence conservation between structurally and functionallyrelated proteins, the amount of sequence identity required to retain afunction or activity (e.g., binding) depends upon the protein, theregion and the function or activity of that region.

The term “identity” and “homology” and grammatical variations thereofmean that two or more referenced entities are the same. Thus, where twosequences are identical, they have the same amino acid sequence. “Areas,regions or domains of identity” mean that a portion of two or morereferenced entities are the same. Thus, where two sequences areidentical or homologous over one or more sequence regions, they shareidentity in these regions.

The extent of identity between two sequences can be ascertained using acomputer program and mathematical algorithm known in the art. Suchalgorithms that calculate percent sequence identity (homology) generallyaccount for sequence gaps and mismatches over the comparison region. Forexample, a BLAST (e.g., BLAST 2.0) search algorithm (see, e.g., Altschulet al., J. Mol. Biol. 215:403 (1990), publicly available through NCBI)has exemplary search parameters as follows: Mismatch −2; gap open 5; gapextension 2. For polypeptide sequence comparisons, a BLASTP algorithm istypically used in combination with a scoring matrix, such as PAM100, PAM250, BLOSUM 62 or BLOSUM 50. FASTA (e.g., FASTA2 and FASTA3) and SSEARCHsequence comparison programs are also used to quantitate the extent ofidentity (Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444 (1988);Pearson, Methods Mol Biol. 132:185 (2000); and Smith et al., J. Mol.Biol. 147:195 (1981)). Programs for quantitating protein structuralsimilarity using Delaunay-based topological mapping have also beendeveloped (Bostick et al., Biochem Biophys Res Commun. 304:320 (2003)).

Modified polypeptide agents also include one or more D-amino acidssubstituted for L-amino acids (and mixtures thereof), structural andfunctional analogues, for example, peptidomimetics having synthetic ornon-natural amino acids or amino acid analogues and derivatized forms.For example, a polypeptide can have 1 to 5, 5 to 10, 10 to 15, 15 to 20,20 to 25, 25 to 30, 30 to 35, 35 to 50, 50 to 100 L or D amino acidresidues, or a combination of L- and D-amino acids or more amino acidresidues.

Modifications include cyclic structures such as an end-to-end amide bondbetween the amino and carboxy-terminus of the molecule or intra- orinter-molecular disulfide bond. Polypeptides may be modified in vitro orin vivo, e.g., post-translationally modified to include, for example,sugar residues, phosphate groups, ubiquitin, fatty acids, lipids, etc.Polypeptide non-amide chemical bonds include, for example, those formedwith glutaraldehyde, N-hydroxysuccinimide esters, bifunctionalmaleimides, N, N′-dicyclohexylcarbodiimide (DCC), ketomethylene,aminomethylene, olefin, ether, thioether and the like (see, e.g.,Spatola in Chemistry and Biochemistry of Amino Acids, Peptides andProteins, Vol. 7, pp 267-357 (1983), “Peptide and BackboneModifications,” Marcel Decker, NY).

Polypeptides and peptidomimetics can be produced and isolated usingmethods known in the art. Peptides can be synthesized, whole or in part,using chemical methods known in the art (see, e.g., Caruthers (1980).Nucleic Acids Res. Symp. Ser. 215; Horn (1980); and Banga, A. K.,Therapeutic Peptides and Proteins, Formulation, Processing and DeliverySystems (1995) Technomic Publishing Co., Lancaster, Pa.). Peptidesynthesis can be performed using various solid-phase techniques (see,e.g., Roberge Science 269:202 (1995); Merrifield, Methods Enzymol.289:3(1997)) and automated synthesis may be achieved, e.g., using theABI 431A Peptide Synthesizer (Perkin Elmer) in accordance with themanufacturer's instructions. Peptides and peptidomimetics can also besynthesized using combinatorial methodologies. Synthetic residues andpolypeptides incorporating mimetics can be synthesized using a varietyof procedures and methodologies known in the art (see, e.g., OrganicSyntheses Collective Volumes, Gilman, et al. (Eds) John Wiley & Sons,Inc., NY). Modified peptides can be produced by chemical modificationmethods (see, for example, Belousov, Nucleic Acids Res. 25:3440 (1997);Frenkel, Free Radic. Biol. Med. 19:373 (1995); and Blommers,Biochemistry 33:7886 (1994).

An agent as defined herein is detectable. An agent can be labeled inorder to be detectable. A label or a tag can be used to provide an agentthat is detectable. Detectable labels include labels suitable fordiagnosis, detection, screening and imaging. A detectable label can beattached to (e.g., linked conjugated) an agent, or be within or be oneor more atoms that comprise the agent. As the structure of agents caninclude one or more of carbon, hydrogen, nitrogen, oxygen, sulfur,phosphorous, etc., radioisotopes of any of carbon, hydrogen, nitrogen,oxygen, sulfur, phosphorous, etc., can be included within an agentdetectably labelled.

Non-limiting exemplary detectable labels also include a radioactivematerial, such as a radioisotope, a metal or a metal oxide.Radioisotopes include radionuclides emitting alpha, beta or gammaradiation. In diagnostic, screening, detection and imaging methods,typically a beta emitter is employed. In particular embodiments, aradioisotope can be one or more of: C, N, O, H, S, Cu, Fe, Ga, Ti, Sr,Y, Tc, In, Pm, Gd, Sm, Ho, Lu, Re, At, Bi or Ac. In additionalembodiments, a radioisotope can be one or more of: ³H, ¹⁰B, ¹⁸F, ¹¹C,¹⁴C, ¹³N, ¹⁸O, ¹⁵O, ³²P, ³⁵S, ³⁵Cl, ⁴⁵Ti, ⁴⁶Sc, ⁵¹Cr, ⁵²Fe, ⁵⁹Fe, ⁵⁷Co,⁶⁰Cu, ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁷⁶Br, ⁷⁷Br, ^(81m)Kr,⁸²Rb, ⁸⁵Sr, ⁸⁹Sr, ⁸⁶Y, ⁹⁰Y, ⁹⁵Nb, ^(94m)Tc, ^(99m)Tc, ⁹⁷Ru, ¹⁰³Ru,¹⁰⁵Rh, ¹⁰⁹Cd, ¹¹¹In, ¹¹³Sn, ^(113m)In, ¹¹⁴In, ¹⁴⁰La, ¹⁴¹Ce, ¹⁴⁹Pm,¹⁵³Gd, ¹⁵⁷Gd, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁶⁹Er, ¹⁶⁹Y, ¹⁷⁵Yb, ¹⁷⁷Lu,¹⁸⁶Re, ¹⁸⁸Re, ²⁰¹Tl, ²⁰³Pb, ²¹¹At, ²¹²Bi or ²²⁵Ac.

Additional non-limiting exemplary detectable labels include a metal or ametal oxide. In particular embodiments, a metal or metal oxide is one ormore of: gold, silver, copper, boron, manganese, gadolinium, iron,chromium, barium, europium, erbium, praseodynium, indium, or technetium.In additional embodiments, a metal oxide includes one or more of:Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III), Pr(III),Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), orEr(III). Metals and oxides include crystals.

Further non-limiting exemplary detectable labels include contrast agents(e.g., gadolinium; manganese; barium sulfate; an iodinated ornoniodinated agent; an ionic agent or nonionic agent); magnetic andparamagnetic agents (e.g., iron-oxide chelate); nanoparticles; an enzyme(horseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase); a prosthetic group (e.g., streptavidin/biotin andavidin/biotin); a fluorescent material (e.g., umbelliferone,fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin); aluminescent material (e g, luminol); or a bioluminescent material (e.g.,luciferase, luciferin, aequorin). A label can be any imaging agent thatcan be employed for detection, screening, diagnostis, or imaging (e.g.,for computed axial tomography (CAT or CT), fluoroscopy, single photonemission computed tomography (SPECT) imaging, optical imaging, positronemission tomography (PET), magnetic resonance imaging (MRI), gammaimaging).

A detectable label can also be linked or conjugated (e.g., covalently)to the agent. In various embodiments a detectable label, such as aradionuclide or metal or metal oxide can be bound or conjugated to theagent, either directly or indirectly. A linker or an intermediaryfunctional group can be used to link an agent to a detectable label.

Linkers include amino acid or peptidomimetic sequences inserted betweenan agent and a label so that the two entities maintain, at least inpart, a distinct function or activity. Linkers may have one or moreproperties that include a flexible conformation, an inability to form anordered secondary structure or a hydrophobic or charged character whichcould promote or interact with either domain Amino acids typically foundin flexible protein regions include Gly, Asn and Ser. Other near neutralamino acids, such as Thr and Ala, may also be used in the linkersequence. The length of the linker sequence may vary withoutsignificantly affecting a function or activity.

Linkers further include chemical moieties, conjugating agents, andintermediary functional groups. Such linkers are often used to bindradioisotopes which exist as, for example, metallic ions (e.g, cations)that bind to groups on the agent. Examples include moieties that reactwith free or semi-free amines, oxygen, sulfur, hydroxy or carboxygroups. Such functional groups therefore include mono and bifunctionalcrosslinkers, such as sulfo-succinimidyl derivatives (sulfo-SMCC,sulfo-SMPB), in particular, disuccinimidyl suberate (DSS), BS3(Sulfo-DSS), disuccinimidyl glutarate (DSG) and disuccinimidyl tartrate(DST). Non-limiting examples include diethylenetriaminepentaacetic acid(DTPA) and ethylene diaminetetraacetic acid (EDTA).

Biological samples include any sample capable of having a biologicalmaterial. Specific non-limiting examples of biological samples includemucus, saliva, feces, blood, serum, plasma, cerebrospinal fluid, urine,or placenta. Biological samples also include biopsies, such as a cell,tissue or an organ biopsy, for example. Non-limiting samples includingbiopsies are of skin, dermis, breast, genito-urinary tract (uterus,ovary, endometrium, vagina, cervix, fallopian tube, bladder, testicle,penis, urinary tract, prostate), lung, nasopharynx, nose or sinuses,thyroid, head, neck, adrenal gland, thyroid, lymph, gastrointestinaltract (stomach, intestine, colon), kidney, pancreas, adrenal gland,liver, bone, bone marrow, heart, muscle, or a sample of the hematopoeticsystem.

A biological sample typically includes cells or cellular material. Cellsthat express a receptor, such as a hormone receptor, for example, a sexor gonadal steroid hormone receptor that can be targeted by an agent inaccordance with methods of the invention also include reproductivecells, tissues and organs, such as breast, ovarian, uterine, cervical,endometrial, prostate, and testicular cells, tissues and organs, as wellas non-reproductive cells, tissues and organs, such as pancreas, skin,liver, colon, kidney, esophagus, brain, blood cells, adrenal, pituitarygland, etc.

In methods of the invention, where a certain amount of expression of areceptor (e.g., a hormone receptor) is predictive or correlates with thepresence of a hyperproliferative disorder (e.g., a tumor, cancer orneoplasia, or metastasis thereof), or that is predictive or correlateswith therapeutic efficacy of a hyperproliferative disorder (e.g., atumor, cancer or neoplasia, or metastasis thereof) treatment or therapy,the cells of the disorder typically express greater amounts of thereceptor (e.g., hormone receptor) than normal cells. For example, cellsof a hyperproliferative disorder (e.g., a tumor, cancer or neoplasia, ormetastasis thereof) may express a hormone receptor that is not typicallydetectably expressed by normal comparison cells. Alternatively, or inaddition, cells of a hyperproliferative disorder (e.g., a tumor, canceror neoplasia, or metastasis thereof) may express greater amounts of areceptor (e.g., hormone receptor) than the amount of receptor that istypically expressed by normal comparison cells. In either case,expression of a receptor not normally expressed in normal cells, orwhere amounts of the receptor are greater than in normal cells, canreveal the presence of a hyperproliferative disorder (e.g., a tumor,cancer or neoplasia, or metastasis thereof) thereby identifying asubject that will or is likely to respond to a given treatment ortherapy that targets a receptor (e.g., treatment of a hyperproliferativedisorder), or correlate with and therefore be predictive of therapeuticefficacy of a hyperproliferative disorder (e.g., a tumor, cancer orneoplasia, or metastasis thereof) treatment or therapy.

In particular non-limiting aspects, to identify a subject that will oris likely to respond to a given treatment or therapy that targets areceptor (e.g., treatment of a hyperproliferative disorder), or for aprediction or correlation with therapeutic efficacy of treatment, atleast 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of thecells express a hormone receptor (e.g., LHRH- or hCG/LH receptors), thannormal cells. In additional particular non-limiting aspects, for aprediction or correlation with therapeutic efficacy, expression of ahormone receptor (e.g., LHRH- or hCG/LH receptors) is at least 25%, 30%,40%, 50%, 60%, 70%, 80%, 90% more than normal cells (i.e., the cellsexpress at least 50% more of a hormone receptor, such as a LHRH- orhCG/LH receptor, than control cells).

Treatments whose therapeutic efficacy can be predicted based uponreceptor expression include LHRH agonists (such as leuprolide,goserelin, buserelin, triptorelin, histrelin and the like) or LHRHantagonists (such as Abarelix, Cetrorelix, Ganirelix, Fe 200486(Degarelix), Teverelix, Elagolix, Antide and the like) and hCG/LHreceptor antisense nucleic acids, polypeptides that bind to LHRH- orhCG/LH receptors and fusion proteins (chimera) that contain LHRH orhCG/LH or their fragments.

Treatments whose therapeutic efficacy can also be predicted based uponreceptor expression include a treatment with a chimera that targetsLHRH- or hCG/LH receptors, for example, a chimera that includes a firstsequence that binds to LHRH- or hCG/LH receptors, and a second cytotoxicmoiety conjugated to said first sequence (e.g., a lytic peptidesequence, a radioisotope, ricin, diphtheria, gelonin, or granzyme B).

A “hyperproliferative cell” or “hyperproliferative disorder” refers toany undesirable or aberrant cell survival (e.g., failure to undergo celldifferentiation, programmed cell death or apoptosis), growth orproliferation. Such disorders include benign hyperplasias,non-metastatic and metastatic neoplasias, cancers, tumors andmalignancies. Undesirable or aberrant cell proliferation andhyperproliferative disorders can affect any cell, tissue, organ in asubject. Undesirable or aberrant cell proliferation andhyperproliferative disorders can be present in a subject, locally,regionally or systemically. A hyperproliferative disorder can arise froma multitude of tissues and organs, including but not limited toreproductive tissues and organs, breast, genito-urinary tract (uterus,ovary, vagina, cervix, endometrium, fallopian tube, bladder, testicle,penis, prostate), lung (e.g., small cell or non-small cell), thyroid,head and neck, brain, nasopharynx, throat, nose or sinuses, lymphoid,adrenal gland, pituitary gland, thyroid, lymph, gastrointestinal (mouth,esophagus, stomach, duodenum, ileum, jejunum (small intestine), colon,rectum), kidney, pancreas, liver, bone, bone marrow, lymph, blood,muscle, skin, and stem cells, which may or may not metastasize to othersecondary sites, regions or locations.

The terms “tumor,” “cancer” and “neoplasia” are used interchangeably andrefer to a cell or population of cells whose growth, proliferation orsurvival is greater than growth, proliferation or survival of a normalcounterpart cell, e.g. a cell proliferative, hyperproliferative ordifferentiative disorder. Typically, the growth is uncontrolled. Theterm “malignancy” refers to invasion of nearby tissue. The term“metastasis” or a secondary, recurring or recurrent tumor, cancer orneoplasia refers to spread or dissemination of a tumor, cancer orneoplasia to other sites, locations or regions within the subject, inwhich the sites, locations or regions are distinct from the primarytumor or cancer.

Invention methods include diagnosing, detecting, screening for, orimaging a primary neoplasia, tumor cancer and metastasis thereof.Metastasis include spreading to other sites, or the formation orestablishment of neoplasia, tumors or cancers at other sites distal fromthe primary neoplasia, tumor or cancer. Thus, methods of the inventioninclude, among other things, diagnosing, detecting, screening for, orimaging a metastases arising from a primary neoplasia, tumor or cancerto one or more other sites, locations or regions distinct from theprimary neoplasia, tumor or cancer, growth or proliferation of ametastasis at one or more other sites, locations or regions distinctfrom the primary neoplasia, tumor or cancer, and formation orestablishment of additional metastasis.

Neoplasias, tumors and cancers that can be diagnosed, detected, screenedfor, or imaged include sarcoma, carcinoma, adenocarcinoma, melanoma,myeloma, blastoma, glioma, lymphoma or leukemia. Exemplary cancersinclude, for example, carcinoma, sarcoma, adenocarcinoma, melanoma,neural (blastoma, glioma), mesothelioma and reticuloendothelial,lymphatic or haematopoietic neoplastic disorders (e.g., myeloma,lymphoma or leukemia).

Neoplasia, tumors and cancers include benign, malignant, metastatic andnon-metastatic types, and include any stage (I, II, III, IV or V) orgrade (G1, G2, G3, etc.) of neoplasia, tumor, or cancer, or a neoplasia,tumor, cancer or metastasis that is progressing, worsening, stabilizedor in remission.

A “solid neoplasia, tumor or cancer” refers to neoplasia, tumor orcancer (e.g., metastasis) that typically aggregates together and forms amass. Specific examples include visceral tumors such as melanomas,breast, pancreatic, uterine and ovarian cancers, testicular cancer,including seminomas, gastric or colon cancer, hepatomas, adrenal, renaland bladder carcinomas, lung, head and neck cancers and braintumors/cancers.

Carcinomas refer to malignancies of epithelial or endocrine tissue, andinclude respiratory system carcinomas (lung, small cell lung),gastrointestinal system carcinomas, genito-urinary system carcinomas,testicular carcinomas, breast carcinomas, prostatic carcinomas,endocrine system carcinomas, and melanomas. The term also includescarcinosarcomas, e.g., which include malignant tumors composed ofcarcinomatous and sarcomatous tissues. Adenocarcinoma includes acarcinoma of a glandular tissue, or in which the tumor forms a glandlike structure. Melanoma refers to malignant tumors of melanocytes andother cells derived from pigment cell origin that may arise in the skin,dermis, eye (including retina), or other regions of the body. Additionalcarcinomas can form from the uterine/cervix, endometrium, lung,head/neck, colon, pancreas, testes, adrenal gland, kidney, esophagus,stomach, liver and ovary.

Sarcomas refer to malignant tumors of mesenchymal cell origin. Exemplarysarcomas include for example, lymphosarcoma, liposarcoma, osteosarcoma,chondrosarcoma, leiomyosarcoma, rhabdomyosarcoma and fibrosarcoma.

Neural neoplasias include glioma, glioblastoma, meningioma,neuroblastoma, retinoblastoma, astrocytoma, oligodendrocytoma

Specific non-limiting examples of neoplasias, tumors and cancers includemalignant and non-malignant neoplasias, tumors and cancers, andmetastasis. In particular, a neoplasia, tumor, cancer or metastasis ofany stage (e.g., stages IA, IB, IIA, IIB, IIIA, IIIB or IV) or grade(e.g., grades G1, G2 or G3).

A “liquid neoplasia, tumor or cancer” refers to a neoplasia, tumor orcancer of the reticuloendothelial or hematopoetic system, such as alymphoma, myeloma, or leukemia, or a neoplasia that is diffuse innature. Particular examples of leukemias include acute and chroniclymphoblastic, myeolblastic and multiple myeloma. Typically, suchdiseases arise from poorly differentiated acute leukemias, e.g.,erythroblastic leukemia and acute megakaryoblastic leukemia. Specificmyeloid disorders include, but are not limited to, acute promyeloidleukemia (APML), acute myelogenous leukemia (AML) and chronicmyelogenous leukemia (CML); lymphoid malignancies include, but are notlimited to, acute lymphoblastic leukemia (ALL), which includes B-lineageALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Specific malignant lymphomasinclude, non-Hodgkin lymphoma and variants, peripheral T cell lymphomas,adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL),large granular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease.

A tumor, cancer or neoplasia, or a metastasis thereof may arise from ormay affect any part of the body of a subject. Exemplary parts (e.g.,organ, tissue) affected include reproductive organs and tissues, skin,dermis, breast, lung, nasopharynx, nose or sinuses, thyroid, head, neck,brain, spine, adrenal gland, thyroid, lymph, blood, gastrointestinal(mouth, esophagus, stomach, duodenum, ileum, jejunum (small intestine),colon, rectum), genito-urinary tract (uterus, ovary, endometrium,vagina, cervix, fallopian tube, bladder, testicle, penis, urinary tract,prostate), kidney, pancreas, adrenal gland, liver, bone, bone marrow,heart, muscle, and the hematopoetic system. Thus, a method of theinvention may be performed to diagnose, detect, screen, or image atumor, cancer or neoplasia, or a metastasis thereof in the whole body ofa subject, a particular region or general area, a specific organ ortissue, or a local portion of a region, organ or tissue.

Methods of the invention include detecting the type, kind, presence orabsence, progression, regression or response, location or extent of atumor, cancer or neoplasia, or a metastasis thereof. Such methods can beused to alternatively or additionally provide information on severity orprogression of a tumor, cancer or neoplasia, or a metastasis thereof;prognosis of a tumor, cancer or neoplasia, or a metastasis thereof;and/or therapy or treatment of a tumor, cancer or neoplasia, or ametastasis thereof based upon detecting, diagnosing, screening orimaging.

As used herein, a “sufficient amount” or “effective amount” or an“amount sufficient” or an “amount effective” refers to an amount thatis, in single or multiple doses, alone or in combination, with one ormore other compositions, sufficient to bind to the receptor therebydetecting a hyperproliferative disorder (e.g., a tumor, cancer orneoplasia, or metastasis thereof). Thus, an amount sufficient oreffective is that amount to allow diagnosis, screening, or imaging of ahyperproliferative disorder (e.g., a tumor, cancer or neoplasia, ormetastasis thereof), identifying a subject that will respond to atherapy or treatment that targets a receptor (e.g., a hyperproliferativedisorder such as a tumor, cancer or neoplasia, or metastasis thereof),or predicting therapeutic efficacy of a given treatment that targets areceptor, such as a hormone receptor, or that targets ahyperproliferative disorder (e.g., a tumor, cancer or neoplasia, ormetastasis thereof).

Methods of detection, diagnostics or screening, such as in vitro, exvivo, and vivo imaging methods, permit the detection of an agent. Suchmethods of detection include magnetic resonance spectroscopy (MRS),magnetic resonance imaging (MRI), positron-emission tomography (PET),gamma-scintigraphy, computed tomography (CT), Computed Axial Tomography(CAT), or single photon emission tomography (SPECT).

Methods also include detecting or diagnosing a subject having or at riskof having a tumor, cancer or neoplasia, (in vivo, ex vivo or in vitro).Such methods include contacting a biological sample from a subject withan agent under conditions whereby the agent can bind to a receptor orcell in the sample, and detecting the agent in the sample to ascertainthe presence or absence or amount of a receptor or hyperproliferativedisorder (e.g., a tumor, cancer or neoplasia, or metastasis thereof), inthe sample, thereby detecting the hyperproliferative disorder (e.g., atumor, cancer or neoplasia, or metastasis thereof), or screening for ordiagnosing the subject as having or not having the hyperproliferativedisorder (e.g., a tumor, cancer or neoplasia, or metastasis thereof), orimaging a hyperproliferative disorder (e.g., a tumor, cancer orneoplasia, or metastasis thereof).

For in vitro, ex vivo, and in vivo diagnosing, detecting, screening orimaging, the type of detection instrument available can depend upon agiven label or conjugate. As an example, a radioisotope or paramagneticisotope is suitable for in vivo detection, diagnosis, screening orimaging. The type of label, such as a radionuclide or metal, will guidethe selection of the instrument used. For instance, decay parameters ofa chosen alpha, beta, or gamma radionuclide chosen can be detectable ormeasured by the selected instrument.

A “subject” refers to an animal, typically mammalian animals, such asbut not limited to non-human primates (apes, gibbons, gorillas,chimpanzees, orangutans, macaques), domestic animals (dogs and cats), afarm animals (chickens, ducks, horses, cows, goats, sheep, pigs),experimental animal (mouse, rat, rabbit, guinea pig) and humans.Subjects include animal disease models, for example, animal models ofundesirable or aberrant cell proliferation, such as a hyperproliferativedisorder (e.g., a metastatic or non-metastatic tumor, cancer, malignancyor neoplasia) for analysis in vivo. Subjects include naturally occurringor non-naturally occurring mutated or non-human genetically engineered(e.g., transgenic or knockout) animals.

Subjects can be any age. For example, a subject (e.g., human) can be anewborn, infant, toddler, child, teenager, or an adult.

Subjects include those in need of a method of the invention, e.g., inneed of diagnosis, detection, screening, imaging, or identifying orpredicting. A subject is considered to be in need of a method of theinvention where a method is likely to provide information concerning thepresence or absence of, the extent or severity of, the location, thestatus or prognosis of, or possible treatment or therapy of ahyperproliferative disorder (e.g., a metastatic or non-metastatic tumor,cancer, malignancy or neoplasia).

Subjects appropriate for methods of the invention therefore includethose having or at risk of having a hyperproliferative disorder (e.g., atumor, cancer or neoplasia, or a metastasis thereof). At risk subjectsinclude subjects that are at risk of developing a hyperproliferativedisorder (e.g., a tumor, cancer or neoplasia, or a metastasis thereof),due to a genetic predisposition or family history, or environmental riskdue to smoking, exposure to smoke or carcinogens, chemicals, sunexposure, etc. A subject may therefore be symptomatic or asymptomaticfor a hyperproliferative disorder (e.g., a tumor, cancer or neoplasia,or a metastasis thereof). The invention methods are therefore applicableto a subject who is at risk of a hyperproliferative disorder (e.g., atumor, cancer or neoplasia, or a metastasis thereof), but has not yetbeen diagnosed for a hyperproliferative disorder (e.g., a tumor, canceror neoplasia, or a metastasis thereof).

Subjects appropriate for treatment include those having or at risk ofhaving a metastatic or non-metastatic tumor, cancer, malignant orneoplastic cell, those undergoing as well as those who are undergoing orhave undergone anti-proliferative (e.g., metastatic or non-metastatictumor, cancer, malignancy or neoplasia) therapy, including subjectswhere the tumor is in remission. “At risk” subjects typically have riskfactors associated with undesirable or aberrant cell proliferation,development of hyperplasia (e.g., a tumor).

Particular examples of at risk or candidate subjects include those withcells that a receptor not typically expressed by normal cells, or atlevels above normal. An agent can bind to such cells selectively,thereby revealing information on the size, severity, prognosis,location, and predicted treatment therapy and outcome. At risk subjectsalso include those that are candidates for and those that have undergonesurgical resection, chemotherapy, immunotherapy, ionizing or chemicalradiotherapy, local or regional thermal (hyperthermia) therapy. Theinvention is therefore applicable to treating a subject who is at riskof a metastatic or non-metastatic tumor, cancer, malignancy or neoplasiaor a complication associated with a metastatic or non-metastatic tumor,cancer, malignancy or neoplasia, for example, due to metastatic ornon-metastatic tumor, cancer, malignancy or neoplasia re-appearance orregrowth following a period of stability or remission.

Risk factors include gender, lifestyle (diet, smoking), occupation(medical and clinical personnel, agricultural and livestock workers),environmental factors (carcinogen exposure), family history (autoimmunedisorders, diabetes, etc.), genetic predisposition, etc. For example,subjects at risk for developing melanoma include excess sun exposure(ultraviolet radiation), fair skin, high numbers of naevi (dysplasticnevus), patient phenotype, family history, or a history of a previousmelanoma. Subjects at risk for developing cancer can therefore beidentified by lifestyle, occupation, environmental factors, familyhistory, and genetic screens for tumor associated genes, gene deletionsor gene mutations. Subjects at risk for developing breast cancer lackBrca1, for example. Subjects at risk for developing colon cancer haveearly age or high frequency polyp formation, or deleted or mutated tumorsuppressor genes, such as adenomatous polyposis coli (APC), for example.

Detectable agents can be incorporated into pharmaceutical compositionsor formulations. Such pharmaceutical compositions/formulations areuseful for administration to a subject, in vivo or ex vivo.

Pharmaceutical compositions and formulations include carriers orexcipients for administration to a subject. As used herein the terms“pharmaceutically acceptable” and “physiologically acceptable” mean abiologically compatible formulation, gaseous, liquid or solid, ormixture thereof, which is suitable for one or more routes ofadministration, in vivo delivery or contact. A formulation is compatiblein that it does not destroy activity of an active ingredient therein(e.g., a detectable agent), or induce adverse side effects that faroutweigh any prophylactic or therapeutic effect or benefit.

Pharmaceutical compositions can optionally be formulated to becompatible with a particular mode or route of administration. Exemplaryroutes of administration include administration to a biological fluid ortissue, mucosal cell or tissue (e.g., mouth, buccal cavity, labia,nasopharynx, esophagus, trachea, lung, stomach, small intestine, vagina,rectum, or colon), neural cell or tissue (e.g., ganglia, motor orsensory neurons) or epithelial cell or tissue (e.g., nose, fingers,ears, cornea, conjunctiva, skin or dermis). Thus, pharmaceuticalcompositions include carriers (excipients, diluents, vehicles or fillingagents) suitable for administration to any cell, tissue or organ, invivo, ex vivo (e.g., tissue or organ transplant) or in vitro, by variousroutes and delivery, locally, regionally or systemically.

Detectable agents can be administered and methods may be practiced viasystemic, regional or local administration, by any route. For example,an agent can be administered systemically, regionally or locally,intravenously, orally (e.g., ingestion or inhalation), intramuscularly,intraperitoneally, intradermally, subcutaneously, intracavity,intracranially, transdermally (topical), parenterally, e.g.transmucosally or rectally for detection. Agents and methods of theinvention including pharmaceutical formulations can be administered viaa (micro)encapsulated delivery system or packaged into an implant foradministration.

Pharmaceutical formulations and delivery systems appropriate for theagents and methods of the invention are known in the art (see, e.g.,Remington: The Science and Practice of Pharmacy (2003) 20^(th) ed., MackPublishing Co., Easton, Pa.; Remington's Pharmaceutical Sciences (1990)18^(th) ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996)12^(th) ed., Merck Publishing Group, Whitehouse, N.J.; PharmaceuticalPrinciples of Solid Dosage Forms (1993), Technonic Publishing Co., Inc.,Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001)11^(th) ed., Lippincott Williams & Wilkins, Baltimore, Md.; andPoznansky et al., Drug Delivery Systems (1980), R. L. Juliano, ed.,Oxford, N.Y., pp. 253-315).

Agents can be administered at any duration or frequency. Typically, anagent is administered as a bolus or is administered in multiple doses toprovide detection, diagnosis, screening or imaging.

The invention provides kits including agents, combination compositionsand pharmaceutical compositions/formulations thereof, packaged into asuitable packaging material. In one embodiment, a kit includes packagingmaterial, an agent and instructions. In various aspects, theinstructions are for administering to a subject or contact of the agentwith a sample to diagnose, detect, screen or image a hyperproliferativedisorder (e.g., a tumor, cancer or neoplasia, or a metastasis thereof).

The term “packaging material” refers to a physical structure housing oneor more components of the kit. The packaging material can maintain thecomponents sterilely, and can be made of material commonly used for suchpurposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules,vials, tubes, etc.). A kit can contain a plurality of components, e.g.,two or more agents alone or in combination.

A kit optionally includes a label or insert including a description ofthe components (type, amounts, doses, etc.), instructions for use invitro, in vivo, or ex vivo, and any other components therein. Labels orinserts include “printed matter,” e.g., paper or cardboard, or separateor affixed to a component, a kit or packing material (e.g., a box), orattached to an ampule, tube or vial containing a kit component. Labelsor inserts can additionally include a computer readable medium, such asa disk (e.g., hard disk), optical disk such as CD- or DVD-ROM/RAM, DVD,MP3, magnetic tape, or an electrical storage media such as RAM and ROMor hybrids of these such as magnetic/optical storage media, FLASH mediaor memory type cards.

Labels or inserts can include identifying information of one or morecomponents therein, dose amounts, clinical pharmacology of the activeingredient(s) including mechanism of action, pharmacokinetics andpharmacodynamics. Labels or inserts can include information identifyingmanufacturer, lot numbers, manufacturer location and date, expirationdates.

Labels or inserts can include information on a hyperproliferativedisorder (e.g., a tumor, cancer or neoplasia, or a metastasis thereof)for which a kit component may be used. Labels or inserts can includeinstructions for a clinician or subject for using one or more of the kitcomponents in a method, diagnostic, detection, screening,indentification, or prediction protocol or regimen, including themethods of the invention. Instructions can include amounts of compound,frequency or duration of administration, and instructions for practicingany of the methods described herein. Kits therefore can additionallyinclude labels or instructions for practicing any of the methods of theinvention described herein including detection, diagnosis, screening,imaging or other methods.

Invention kits can additionally include a buffering agent, or apreservative or a stabilizing agent in a formulation containing an agentof the invention. Each component of the kit can be enclosed within anindividual container and all of the various containers can be within asingle package. Invention kits can be designed for cold storage.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or study of the present invention, suitable methods andmaterials are described herein.

All of the features disclosed herein may be combined in any combination.Each feature disclosed in the specification may be replaced by analternative feature serving a same, equivalent, or similar purpose.Thus, unless expressly stated otherwise, disclosed features (e.g.,compound structures) are an example of a genus of equivalent or similarfeatures.

All applications, publications, patents and other references, GenBankcitations and ATCC citations cited herein are incorporated by referencein their entirety. In case of conflict, the specification, includingdefinitions, will control.

As used herein, the singular forms “a”, “and,” and “the” include pluralreferents unless the context clearly indicates otherwise. Thus, forexample, reference to an “agent,” “compound” or “a receptor” includes aplurality of agents/compounds/receptors and reference to a “tumor,”“cancer,” “neoplasia” or “metastasis” thereof can include reference toone tumors, cancers, neoplasias, metastases, and so forth.

As used herein, all numerical values or numerical ranges includeintegers within such ranges and fractions of the values or the integerswithin ranges unless the context clearly indicates otherwise. Thus, toillustrate, reference to a range of 90-100%, includes 91%, 92%, 93%,94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%,etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth. Referenceto a range of 0-72 hrs, includes 1, 2, 3, 4, 5, 6, 7 hrs, etc., as wellas 1, 2, 3, 4, 5, 6, 7 minutes, etc., and so forth. Reference to a rangeof 0-72 hrs, includes 1, 2, 3, 4, 5, 6, 7 hrs, etc., as well as 1, 2, 3,4, 5, 6, 7 minutes, etc., and so forth. Reference to a range of doses,such as 0.1-1 ug/kg, 1-10 ug/kg, 10-25 ug/kg, 25-50 ug/kg, 50-100 ug/kg,100-500 ug/kg, 500-1,000 ug/kg, 1-5 mg/kg, 5-10 mg/kg, 10-20 mg/kg,20-50 mg/kg, 50-100 mg/kg, 100-250 mg/kg, 250-500 mg/kg, includes0.11-0.9 ug/kg, 2-9 ug/kg, 11.5-24.5 ug/kg, 26-49 ug/kg, 55-90 ug/kg,125-400 ug/kg, 750-800 ug/kg, 1.1-4.9 mg/kg, 6-9 mg/kg, 11.5-19.5 mg/kg,21-49 mg/kg, 55-90 mg/kg, 125-200 mg/kg, 275.5-450.1 mg/kg, etc. Aseries of ranges, for example, 1-10 ug/kg, 10-25 ug/kg, 25-50 ug/kg,50-100 ug/kg, 100-500 ug/kg, 500-1,000 ug/kg, 1-5 mg/kg, 5-10 mg/kg,10-20 mg/kg, 20-50 mg/kg, 50-100 mg/kg, 100-250 mg/kg, 250-500 mg/kg,includes 1-25 ug/kg, 10-25 ug/kg, 25-100 ug/kg, 100-1,000 ug/kg, 1-10mg/kg, 1-20 mg/kg etc.

The invention is generally disclosed herein using affirmative languageto describe the numerous embodiments. The invention also includesembodiments in which subject matter is excluded, in full or in part,such as substances or materials, method steps and conditions, protocols,or procedures. Thus, even though the invention is generally notexpressed herein in terms of what the invention does not include aspectsthat are not expressly excluded in the invention are neverthelessdisclosed herein.

A number of embodiments of the invention have been described.Nevertheless, one skilled in the art, without departing from the spiritand scope of the invention, can make various changes and modificationsof the invention to adapt it to various usages and conditions. Forexample, salts, isomers, racemers, multimers of agents disclosed hereinare within the scope of this invention. Accordingly, the followingexamples are intended to illustrate but not limit the scope of theinvention claimed.

EXAMPLES Example 1 This Example Includes Methods Used to Determine LHRH-and LH Receptor Status in Tumor Biopsies. LHRH Receptor Determination

The presence of LHRH receptor was determined in tumor biopsies frompatients diagnosed with specific forms of cancer. In particular, casesof breast cancer, endometrial cancer and ovarian cancer diagnosed andanalyzed included patients who were evaluated in the past foreligibility for Herceptin therapy. A database was established includingage of the patient, TNM/FIGO staging information, estrogen, progesteroneand HER2/neu receptor status for breast cancer cases, recurrence andsurvival information. Cases were reviewed for eligibility based onenough tumor available for analysis. Identifiers were removed from thetissue blocks and were given protocol identification numbers.

The Sequence Listing submitted herewith on compact disc is incorporatedherein by reference.

Immunoperoxidase staining was performed on the tumor biopsy tissue usingthe GnRH(LHRH) receptor mouse monoclonal antibody stain (Clone A9E4)marketed by Vector Laboratories (VP-G811, Lot number 13308, Burlingame,Calif.) at a dilution of 1:30, and the Ventana NIEW DAB Detection kit.The immunoperoxidase stains were performed on one of the Ventana XT,Ventana Benchmark or Ventana Nexus units (Ventana Medical Systems, Inc.Tucson, Ariz.). The immunoperoxidase staining was analyzed utilizing theVentana Image Analysis System, an adjunctive computer-assisted imageanalysis system functionally connected to an interactive microscope(Axio Imager, Carl Zeiss, NY). The quantitative analysis was performedutilizing the program for quantitation of the HER2/neu receptor whichincludes morphometric and colorimetric analysis.

Receptor status results were reported as 0, 1+, 2+ or 3+. In addition, amanual assessment of staining was performed using the followingcriteria: Non-immunoreactive; Immunoreactive, Score 1+: 1-25% positivecells; Immunoreactive, Score 2+: 26-50% positive cells; Immunoreactive,Score 3+: 51-75% positive cells; Immunoreactive, Score 4+: 76-100%positive cells. Assessment of strength of staining was added to themanual evaluation using the following criteria: 0: Negative; 1: Weak,only visible at high magnification; 2: Moderate staining visible at lowmagnification; 3: Strong staining visible at low magnification. The IHCfigures list 0, +1, +2, or +3 as the particular score, which reflectsthe relative immunoreactive receptor present.

LH Receptor Determination

The presence of LH receptor was determined in tumor biopsies frompatients diagnosed with specific forms of cancer (as described above).Immunoperoxidase staining of tumor biopsy tissues was performed usingthe rabbit anti-human polyclonal antibody targeting the C-terminaldomain, (MBL Laboratories, Woburn Mass.), diluted at 1:30 and theVentana NIEW DAB Detection kit. The immunoperoxidase stains wereperformed on one of the Ventana XT, Ventana Benchmark or Ventana Nexusunits (Ventana Medical Systems, Inc. Tucson, Ariz.). Theimmunoperoxidase staining was analyzed utilizing the Ventana ImageAnalysis System, an adjunctive computer-assisted image analysis systemfunctionally connected to an interactive microscope (Axio Imager, CarlZeiss, NY). The quantitative analysis was performed utilizing theprogram for quantitation of the HER2/neu receptor which includesmorphometric and colorimetric analysis.

Receptor status results were reported as 0, 1+, 2+ or 3+. In addition, amanual assessment of staining was performed using the followingcriteria: Non-immunoreactive; Immunoreactive, Score 1+: 1-25% positivecells; Immunoreactive, Score 2+: 26-50% positive cells; Immunoreactive,Score 3+: 51-75% positive cells; and, Immunoreactive, Score 4+: 76-100%positive cells. Assessment of strength of staining was added to themanual evaluation using the following criteria: 0: Negative; 1: Weak,only visible at high magnification; 2: Moderate staining visible at lowmagnification; and, 3: Strong staining visible at low magnification.

Example 2 This Example Includes Receptor Expression Profiles of VariousHuman Tumors.

Analysis of ovarian, endometrial and breast cancer tumors was performedaccording to the methods of Example 1.

Receptor Expression Profile in Ovarian Cancers

Receptor profiles were determined in 50 samples of ovarian cancer basedupon the analysis of tissues from biopsies of primary tumors (1°biopsy), and recurrences. Of the 50 biopsies studied, 82% were positivefor LHRH receptors. In particular, 54-56% graded immunoreactive with ascore 1+, 26-28% graded 2-3+ staining for LHRH receptors, and, 18%graded non-immunoreactive for LHRH receptors with a score of 0. Analysisof the tumor biopsies for LH receptors revealed that, 98% were found tobe positive for LH receptors. In particular, 40-44% gradedimmunoreactive with a score of 1+, 74% graded 2-3+, for LH-receptors,and, 2% graded non-immunoreactive with a score of 0. LH- and LHRHreceptor profiles were also determined in 11 cases of ovarian cancersfrom biopsies of recurrent tumors. Of these 11 cases, 54.5% gradedimmunoreactive with a score of 1+, 27.2% graded 2+, and 9% graded 3+ forLHRH receptors. Analysis of the biopsies of recurrent tumors for LHreceptors revealed that, 27% graded immunoreactive with a score of 1+,54.5% graded 2+, and 18.1% graded 3+ for LH.

Receptor Expression Profile in Endometrial Cancers

Receptor profiles were determined in 50 samples of endometrial cancerrepresenting 40 1° biopsies and 10 metastases. Of the 50 cases studied,80-86% of the biopsies were positive for LHRH receptors. In particular,50-66% graded immunoreactive with a score of 1+, 14-36% graded 2-3+, forLHRH receptors, and, 14-20% graded non-immunoreactive with a score of 0.Analysis of the biopsies for LH receptors revealed that, 96-98% werepositive for LH receptors. In particular, 28-32% graded immunoreactivewith a score of 1+, 64-70% graded 2-3+, for LH receptors, and 2-4%graded non-immunoreactive with a score of 0. LH- and LHRH receptorprofiles were also determined in 10 cases of endometrial cancers frombiopsies of metastases. Of these 10 cases, 40% graded immunoreactivewith a score of 1+, 40% graded 2+, and 10% graded 3+ for LHRH receptors.Analysis of the biopsies of metastases for LH receptors revealed that,10% graded immunoreactive with a score of 1+, 30% graded 2+, and 30%graded 3+ for LH receptors.

Receptor Expression Profile in Breast Cancers

Receptor profiles were determined in 98 samples of breast cancerbiopsies (primary, metastatic and recurrent) Of the 98 biopsies studied,84-87% were positive for LHRH receptors. In particular, 48-57% gradedimmunreactive with a score of 1+, 26-38% graded 2-3+, and 13-26% werenegative (0) for LHRH receptors. Analysis of biopsies for LH receptors,revealed that 99% were positive for LH receptors. In particular, 18-23%graded immunoreactive with a score of 1+, 74% graded 2-3+ and 1% showednegative staining for LHreceptors. In particular, LH- and LHRH receptorprofiles were determined in 22 cases of breast cancer by analysis oftissues taken from biopsies of secondary (2°) or metastatic tumors. Ofthe 22 cases studied, 10% of the biopsies were positive for the LHRHreceptor. In particular, 60% graded immunoreactive with a score of 1+,13% graded 2+, and 15% graded 3+ for LHRH receptors in recurrences. Uponanalysis of the biopsies for LH receptors, 95% were positive for LHreceptor. In particular, 26.3% graded immunoreactive with a score of 1+,47.3% graded 2+, and 31.5% graded 3+ for LH receptors in metastases.

In sum, the foregoing studies reveal that various human cancer tumorsexpress LHRH- and LH receptors at high levels. In particular, LHRH- andLH receptors were expressed at high levels in ovarian, endometrial andbreast tumors including metastases and recurrent tumors. Studies revealthat LHRH- and LH receptor expression is not compromised followingstandard of care treatment. Analysis of metastatic or secondary (2°)tumor biopsies revealed that recurrent tumors express both LHRH- and LHreceptors. Furthermore, receptors can be expressed at higher levels insecondary (2°) tumors compared to levels expressed in primary tumors.For example, LHRH receptors have a greater level of expression inmetastatic or secondary (2°) breast cancer tumors than in primary breasttumors.

Example 3 This Example Includes a Determination of LHRH- and LH ReceptorStatus in Various Human Tumor Cell Lines. Quantification of FunctionalLHRH Receptors in Various Cancer Cell Lines

LHRH receptor presence has been reported for a number of human cancercell lines using immunohistochemistry techniques as well as geneexpression. However, these reports vary even within the same cell linebecause of differences in passage numbers of the cell line studied, andare therefore inconsistent. To detect and quantify the LHRH receptorstatus in human cancer cell lines, procedures and sensitivity of anarray of cell lines were evaluated and quantified for functional LHRHreceptors using immunohistochemistry techniques. The data were thencorrelated with sensitivity to EP-100 (KFAKFAKKFAKFAKKFAKQHWSYGLRPG)(SEQ ID NO.: 1) treatment.

LHRH receptor status was determined using an array of human cancer celllines. Cell cultures were obtained from the American Culture type andTissue Collection and cultured according to standard conditionsdescribed by ATCC. Cell were seeded at a number of 10,000 cells/wellinto each well of glass chamber slide in 4 chamberslide compartments andcultivated in growth medial until confluent. Monolayers were fixed using10% phosphate buffered formalin and preserved in ethanol aftersuccessive dehydration. These chamber slides underwent immunoperoxidasestaining using the mouse monoclonal antibody for LHRH receptors (GNRH03,#MS-1139-P, LabVision). The immunoperoxidase procedure was performed onVentana XT, Ventana Benchmark or Ventana Nexes Units.

Quantitative immunoperoxidase image analysis was conducted with theVentana Image Analysis System (VIAS) adjunctive computer assisted imageanalysis system functionally connected to an interactive microscope(Axio Imager). The quantitative analysis was conducted with the programfor quantification of Her2/neu receptor that included morphometric andcolorimetric analysis. Receptor status results were reported aspercentage of cells showing positive staining of the LHRH receptorsunder the following criteria: 0 non-immunoreactive, 1+: 1-25% positive,2+26-50% positive, 3+51-75% positive cells. These data were compared tomanual assessments of staining strength. The following cell lines wereanalyzed: MDA-MB-435S (p250) (estrogen receptor negative), T47 D (p 100)estrogen receptor alpha positive, MCF-7 (p 152) estrogen receptor alphapositive, MCF-10 A (p 100), PC-3 (p 26) androgen receptor negative, Kle(p 2), Hec-1A (p 10) and 3T3 (p 122), OVCAR-3 (p41) and SKOV-3 (p 40).

The data show that LHRH receptor negative ovarian cancer cell lineSKOV-3 had a VIAS score of (0), and the ovarian cancer cell line OVCAR-3scored (2+). Among breast cancer cell lines T47 D was (1+), MCF-7 (3+),MDA-MB-435S (3+), prostate cancer cell line, PC-3 (2+), endometrialcancer cell lines Kle (0) and Hec 1A (0). Non cancerous cell lines wererepresented by MCF-10 A breast epithelial (0) and 3T3 mouse fibroblastcells (0). Thus, various cancer cell lines present surface LHRHreceptors to varying degrees.

Correlation of In Vitro Cytotoxicity of EP-100 in Human Cancer CellLines with Surface LHRH-Receptor Expression Levels

Studies were designed to test receptor specificity, correlation ofsensitivity to LHRH receptor expression and initial efficacy kinetics invitro by comparing targeted kinetics to untargeted kinetics. Thecytotoxicity of EP-100 (KFAKFAKKFAKFAKKFAKQHWSYGLRPG) (SEQ ID NO.: 1) inhuman cancer cell lines expressing various degrees of LHRH receptors wasdetermined in an array of studies ranging from incubations of 24 hours.Cytotoxicity was evaluated as IC₅₀ value for each cell line. The datawere then correlated to LHRH receptor expression determined in the sameculture through immunohistochemistry surface LHRH receptor staining asVIAS scores to determine specificity of EP-100 targeting.

In vitro toxicity of EP100 was analyzed in human cancer cell lines thatover-express LHRH receptors at various receptor capacities (MDA-MB-4355(575 fmol/mg protein (Chatzistamou et al., Clinical Cancer Res. 6:4158(2000)), VIAS Score+3), MCF-7 (579 fmol/mg protein; VIAS Score 3+) T47 D(VIAS Score 1+), OVCAR-3 (196 fmol/mg protein (Volker et al., American JObstetrics and Gynecology 186:171 (2002)), VIAS Score 2+), LNCaP (355fmol/mg protein) PC-3 (197 fmol/mg protein, Leuschner et al. TheProstate 56:239 (2003), VIAS Score 2+), AN3-CA (IHC positive), Hec-1A(54 fmol/mg protein, VIAS score 0 (Volker et al., American J Obstetricsand Gynecology 186:171 (2002), Chatzaki et al. Cancer Res. 56:2059(1996)) among those two multi-drug resistant cancer cell lines. TheLHRH-receptor negative cell lines SKOV-3 (VIAS Score 0) and Hec 1A wereused as measure for specificity (Westphalen et al., International J.Oncology 17:1063 (2000)).

Cell cultures were prepared in 96 well plates using 10,000 cells/welland were allowed to attach for 48 hours. EP-100 in lyophilized form wasfreshly dissolved in saline and added into the multi-well plates atincreasing concentrations of 0, 0.001, 0.01, 0.1, 1, 2, 5, 10 and 100μM. Incubations were conducted for 24 h at 37° C. Cell viability wasdetermined using formazan conversion assays (MTT assays). Controlscontained USP saline or 0.1% TritonX-100™ as reference for 0 and 100%cell death, respectively.

Data were processed and analyzed using Graph Pad Prizm4™ software (GraphPad Prizm, Inc). Statistical analysis for significance was determined bya two-tailed Student's T-test.

Human cancer cell lines showed variable sensitivities to the drug, whichcorrelated with LHRH receptor capacities. Human breast cancer cell line(MDA-MB-231, negative for estrogen receptors, Her2/neu and progesteronereceptors, multi drug resistant) was insensitive to cisplatinum, butsensitive to EP-100 (5.5 μM), followed by the human endometrial cancercell line AN3-CA (3.3 μM), the ovarian cancer cell line OVCAR-3 (3.0μM), the prostate cancer cell lines LNCaP (1.5 μM) and PC-3 (5.5 μM).The MDA-MB-4355 breast cancer cell line showed highest sensitivity with0.8 μM and has the highest LHRH receptor capacity of all tested celllines (FIGS. 2A and 2B). SKOV-3, a LHRH receptor negative ovarian cancercell line, was insensitive to EP-100 with >10 μM.

In conclusion, human cancer cell lines show high correlation ofsensitivity to EP-100 and their surface LHRH receptor expressiondetermined as LHRH receptor capacities (r²=0.82) as well as LHRHreceptor expression determined and quantified in immunohistochemistrymethods (r²=0.64). The greater LHRH receptor expression on the cellsurface correlates with increased sensitivity to LHRH receptor targetingdrug EP-100.

Example 4 This Example Includes Determination and Quantification ofLH-Receptors and LHRH-Receptors in Human Cancer Cell Lines Through LHRHReceptor and LH/hCG Receptor Gene Expression Assays.

A quantitative human LHRH receptor and LH/hCG receptor gene expressionassay has been developed to identify malignant cell lines and patienttumor specimens. LHRH receptor and LH/hCG receptor mRNA levels inmultiple cancer cell lines were measured to identify potential targetsfor LHRH- and LH/CG receptors. This method determines the LHRH- andLH/CG receptor expression on RNA detection levels.

In brief, RNA was isolated from cell cultures or tissue samples usingthe RNeasy Mini Kit (Qiagen #74104) according to the standard protocolprovided. Contaminating genomic DNA was removed through an on columnDNase digestion step. RNA samples were stored in RNase-free watercontaining RNase inhibitor at −80° C. until analysis.

For each RNA type quantification, reverse transcriptase polymerase chainreaction was conducted using Taqman primers for LHRH receptor and LHreceptor. The LH receptor Set 2 primer/probe was designed to amplifyfull length transcripts containing the transmembrane region of the LHreceptor. RNA was quantified and analyzed for integrity and DNAcontamination using the Agilent 2100 Bioanalyzer, according tomanufacturer instructions. Profiles generated by the Agilent provideconcentration, visual inspection of RNA integrity, and an RNA integritynumber based on the entire electrophoretic trace of a sample.

What is claimed:
 1. A method of analyzing a prostate, ovarian, breast orendometrial sample, the method comprising: (a) contacting the prostate,ovarian, breast or endometrial sample from a subject, with a hormonepolypeptide or hormone polypeptide analog that binds to LHRH- or hCG/LHreceptors under conditions allowing the hormone or hormone analog tobind the LHRH- or hCG/LH receptors; (b) determining an amount of LHRH-or hCG/LH receptors in the sample by detecting the amount of hormonepolypeptide or hormone polypeptide analog that is bound to the LHRH- orhCG/LH receptors in the sample; (c) contacting cells obtained from thesample with a peptide comprising the cytotoxic sequenceKFAKFAKKFAKFAKKFAKQHWSYGLRPG (SEQ ID NO: 1); and (d) after thecontacting of (c), determining the IC50 value according to a sensitivityof the cells to SEQ ID NO:1, wherein the IC50 value is 5.5 μM or lessand wherein greater LHRH- or hCG/LH receptor expression indicatesincreased sensitivity of the cells.
 2. The method of claim 1, comprisingidentifying a sample according to the determining of (b), whereingreater than 50% of the cells express LHRH- or hCG/LH receptors.
 3. Themethod of claim 1, wherein the cytotoxic sequence is biotin-conjugated.4. The method of claim 1, wherein the hormone polypeptide or hormonepolypeptide analog comprises one or more D-amino acids.
 5. The method ofclaim 1, wherein the hormone polypeptide or hormone polypeptide analogis biotin-conjugated.